DGH - Dissertações de mestrado
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- Assessing the pro inflammatory effects of bisphenol compounds using exposure relevant in vitro co culture modelsPublication . Pereira, Gonçalo Alexandre Candeia; Jordan, Peter; Rodrigues, CecíliaInflammation has reached epidemic proportions in industrialized countries, mainly due to unhealthy habits, poor diet, environmental pollution and other factors not yet understood. If uncontrolled or prolonged, inflammation can become chronic and contribute to the development of a number of human diseases, including autoimmune diseases, intestinal diseases and, in the worst cases, tumorigenesis and tumor progression. Exposure to endocrine disrupting chemicals (EDCs) is one environmental factor contributing to inflammation, and recent studies have brought the bisphenol (BP) group of EDCs into the scientific spotlight. They have been strongly linked to various pathologies, including chronic inflammation, and their effect on human gut health is a hot topic in the scientific community. With this in mind, the aim of this work was proposed to analyze the effects of four bisphenols, BPA, BPS-MAE, BPAP and BPP, on intestinal barrier stress and associated pro-inflammatory effects. To achieve this, a co-culture system was optimized and established, consisting of an improved protocol of polarized Caco-2 epithelial cells seeded on PET insert filters in an apical compartment, together with THP-1 derived macrophages in a basolateral compartment. Subsequently, the effects of BPs exposure on barrier integrity, cellular stress and pro-inflammatory cytokine were tested in a wide range of concentrations (from 100 μM to 0.1 μM). Experimentally, we found that the model was capable of delivering BP-specific data on potential health effects. In terms of transepithelial resistance and epithelial stress, we were able to identify some clear trends that need to be consolidated with more independent experimental replicates. In particular, BPA was the least potent inducer of cellular stress responses and changes in epithelial polarization, whereas the BP analogues tested proved to be more disruptive than BPA, with BPP appearing to be the most potentially hazardous, followed by BPAP and then BPS-MAE. To access the inflammation-modulating effects of these compounds, we tested macrophages, either directly or as co-cultured cells, for expression of the pro-inflammatory marker IL-1β using a semiquantitative RT-PCR approach. An important optimization was their priming with IFN-γ to increase the sensitivity of the model and allow for more physiological relevance. Our observations showed that, once again, the BP analogues induced greater effects compared to BPA. BPP appeared to be the more potent inducer of inflammation, followed by BPS-MAE. Both showed elevated levels of the IL-1β marker at all concentrations tested. BPAP and BPA produced more attenuated effects, although significant at higher concentrations. In conclusion, this work has provided us with landmark results on these BPA analogues and their effects on gut health, adding new insights into the 'new generation' of emerging BPs and their potential adverse health effects.
- Impact of BCL-6 downregulation in the oncogenic properties of breast cancer cellsPublication . Jorge, João Miguel Dyson de Lima; Barros, Patrícia; Jordan, PeterBreast cancer (BC) incidence has risen over the past two decades, now being the second most prevalent cancer worldwide and the fourth leading cause of cancer-related deaths. Despite advancements in BC treatment, challenges like acquired resistance, recurrence, and metastasis persist. BCL-6, a transcriptional repressor, plays a controversial role in BC development. It is overexpressed in approximately half of primary tumors across all subtypes, correlating with poorer patient prognosis. Conversely, its downregulation is linked to disease progression and metastasis, highlighting the critical need for a deeper understanding of BCL-6's dual role in BC pathogenesis. This study used RNA interference to explore the impact of BCL-6 depletion on the oncogenic progression of MCF-7 cells, a low-tumorigenic estrogen receptor-alpha-positive (ERα+) cell line. While BCL-6 is known to regulate mammary cell proliferation and differentiation, its depletion did not affect MCF-7 cell proliferation or viability but significantly reduced their individual and collective migratory properties. An RNA microarray analysis identified a set of genes upregulated following BCL-6 depletion, including S100A7, previously reported to inhibit MCF-7 cell migration and invasion in ERα+ BC cells. However, our findings showed that S100A7 downregulation alone did not affect MCF-7 migration. Moreover, simultaneous depletion of BCL-6 and S100A7 failed to restore MCF-7 cell migratory behavior. Our results suggest that increased expression of BCL-6 is linked to increased cell migration but is independent of S100A7 upregulation. Further studies are required to clarify the role of BCL-6 in BC, including disease progression.
- Pesquisa de Deleções/Duplicações em Genes Associados a Cancro Hereditário por MLPA DigitalPublication . Alves, Beatriz Correia; Gonçalves, João; Melo, Maria Joana Lima BarbosaA presença, na linha germinativa, de Variações do Número de Cópias (CNV) em genes de predisposição para cancro hereditário pode aumentar a suscetibilidade a esta doença. A identificação de uma CNV patogénica ou provavelmente patogénica num doente oncológico tem um impacto significativo na gestão clínica do indivíduo afetado e dos seus familiares. Tradicionalmente, a pesquisa de CNV no diagnóstico molecular de cancro hereditário é realizada apenas para alguns genes através do MLPA (Multiplex Ligation-dependent Probe Amplification) convencional. Nos últimos anos, o desenvolvimento de softwares de análise in silico de CNV com base em dados de NGS (Next-Generation Sequencing) representou um avanço significativo, ao possibilitar a pesquisa de deleções e duplicações em múltiplos genes em simultâneo. No entanto, estas ferramentas apresentam ainda limitações. Dada a relevância de uma análise abrangente que integre o maior número possível de genes relevantes no âmbito da patologia em causa, este trabalho teve como principal objetivo implementar uma nova metodologia de pesquisa de CNV em genes associados a cancro hereditário, que combina os princípios do MLPA convencional com a capacidade da NGS de analisar vários genes em simultâneo: o MLPA digital. Neste estudo, foi realizada a pesquisa de CNV por MLPA digital em amostras de doentes com história clínica e familiar de cancro, seguida de validação dos resultados utilizando outras metodologias de genética molecular e classificação das variantes identificadas segundo as recomendações da CanVIG-UK. O MLPA digital demonstrou ser eficaz na deteção de deleções e duplicações em genes associados a cancro hereditário, apresentando um desempenho adequado para a utilização em laboratórios clínicos, com sensibilidade de 100% e especificidade de 98%. A eficácia dos softwares de pesquisa in silico panelcn.MOPS e DRAGEN Enrichment foi confirmada através da concordância entre os resultados destas ferramentas e do MLPA digital. Foram identificadas cinco variantes patogénicas ou provavelmente patogénicas nos genes APC, BRCA1, BRCA2 e CHEK2, que justificam os fenótipos dos doentes. Este estudo demonstra que o MLPA digital é uma alternativa ao MLPA convencional na primeira fase de pesquisa molecular de CNV germinativas em genes associados a cancro hereditário, permitindo a análise de múltiplos genes em várias amostras em simultâneo.
- Regulation of PERK gene expression by its upstream open reading framesPublication . Ponte, João Gonçalves da; Zilhão, Rita; Romão, LuísaUpstream open reading frames (uORFs) are cis-acting elements located within the 5’ untranslated region (5’UTR) of transcripts, which can regulate the translation of the corresponding main open reading frame (mORF). In normal conditions, uORFs are typically repressors of downstream translation, as they can block ribosomal access to the mORF or even induce mRNA degradation through the nonsense-mediated mRNA decay (NMD) pathway. However, in stress conditions, phosphorylation of the eukaryotic initiation factor 2 (eIF2) allows the expression of several stress-responsive proteins via uORF-mediated mechanisms, while global mRNA translation is inhibited. During endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins leads to activation of the ER-resident PKR-like ER kinase (PERK), which will phosphorylate the α-subunit of eIF2 as part of the stress-protective mechanisms of the unfolded protein response (UPR) and integrated stress response (ISR). This results in selective uORF-mediated translation of downstream effectors, which will drive stress resolution or cell death in case of prolonged stress. The dual role of PERK in regulating cell fate is implicated in a growing list of pathophysiological conditions, including neurological and cardiovascular diseases, ophthalmological disorders, viral infections, cancer, and diabetes. Moreover, mutations in the EIF2AK3 gene encoding PERK are implicated in a rare autosomal recessive disorder, the Wolcott-Rallison Syndrome (WRS). Data from ribosome-profiling (Ribo-seq) studies indicate the existence of uORFs within PERK 5’UTR which could be involved in regulating PERK expression. This work aims to study the translational regulatory role of the uORFs identified in PERK’s 5’UTR and estimate its impact on cell homeostasis and human disease. We wish to highlight the importance of including 5’UTRs in the screening of disease-related mutations, as well as the necessity of functional studies to assess their relevance in the pathogenesis of human diseases, as it may provide vital information for developing new therapeutic strategies.
- Repurposing of CFTR modulator drugs in the context of colorectal cancerPublication . Vincente, Luana Pimenta; Matos, Paulo; Jordan, PeterColorectal cancer (CRC) remains one of the leading causes of cancer death worldwide and is considered to arise from genetic and epigenetic changes due to interactions between the tumor and the microenvironment that surrounds it. Recently, the chloride and bicarbonate channel CFTR (cystic fibrosis transmembrane conductance regulator) was implicated in CRC etiology, since a decreased CFTR expression is associated to higher aggressiveness and lower survival in sporadic CRC. Moreover, CFTR mutations are the cause for the autosomal recessive disease cystic fibrosis (CF), and individuals with CF have a higher risk of developing CRC. Several CFTR modulator drugs have been recently clinically approved to improve CFTR functional expression in CF individuals. The current Masters’ project aimed to determine if these drugs can also be used to improve CFTR abundance in non-CF CRC cells, and whether these could be used to reduce their oncogenic properties. For this purpose, four CRC cell lines, with different CFTR expression levels, were cultured and treated with three CFTR modulator drugs, individually and in combination. It was observed that the combination treatment with the three drugs significantly increased CFTR protein levels in the Caco-2 and DLD-1 cells, but had no significant impact in either HCT116 or HT29 cells, likely due to their particular genetic and epigenetic backgrounds. Importantly, treatment with CFTR modulators significantly inhibited migration of Caco-2 and DLD-1 cells, without markedly impacting their viability. These findings suggest that CFTR modulators have potential as therapeutic agents to counteract the oncogenic properties of CRC cells with specific genetic and epigenetic profiles. However, the impact of CFTR modulator treatment in CRC development will need to be validated in vivo using mouse models. Repurposing these modulators for CRC treatment could enhance outcomes for CRC patients while also reducing costs for CF patients by expanding the clinical applications of these drugs.